J Vet Intern Med 2014;28:1770–1774

Pituitary Dwarfism in Saarloos and Czechoslovakian Wolfdogs is Associated with a Mutation in LHX3 A.M.W.Y. Voorbij, P.A. Leegwater, and H.S. Kooistra Background: Pituitary dwarfism in German Shepherd Dogs is associated with autosomal recessive inheritance and a mutation in LHX3, resulting in combined pituitary hormone deficiency. Congenital dwarfism also is encountered in breeds related to German Shepherd Dogs, such as Saarloos and Czechoslovakian wolfdogs. Objectives: To investigate whether Saarloos and Czechoslovakian wolfdog dwarfs have the same LHX3 mutation as do Germans Shepherd Dog dwarfs. A specific aim was to determine the carrier frequency among Saarloos and Czechoslovakian wolfdogs used for breeding. Animals: Two client-owned Saarloos wolfdogs and 4 client-owned Czechoslovakian wolfdogs with pituitary dwarfism, 239 clinically healthy client-owned Saarloos wolfdogs, and 200 client-owned clinically healthy Czechoslovakian wolfdogs. Methods: Genomic DNA was amplified using polymerase chain reaction (PCR). In the Saarloos and Czechoslovakian wolfdog dwarfs, PCR products were analyzed by sequencing. DNA fragment length analysis was performed on the samples from the clinically healthy dogs. Results: Saarloos and Czechoslovakian wolfdog dwarfs have the same 7 bp deletion in intron 5 of LHX3 as do German Shepherd Dog dwarfs. The frequency of carriers of this mutation among clinically healthy Saarloos and Czechoslovakian wolfdogs used for breeding was 31% and 21%, respectively. Conclusions and Clinical Importance: An LHX3 mutation is associated with pituitary dwarfism in Saarloos and Czechoslovakian wolfdogs. The rather high frequency of carriers of the mutated gene in the 2 breeds emphasizes the need for screening before breeding. If all breeding animals were genetically tested for the presence of the LHX3 mutation and a correct breeding policy would be implemented, this disease could be eradicated completely. Key words: Canine; Genetic testing; Growth hormone; Mutation.

evelopment of the adenohypophysis is a very complicated process and any defect in the development of this gland may result in a form of isolated or combined pituitary hormone deficiency (CPHD). In dogs, pituitary dwarfism is the most common manifestation of CPHD and this disorder is encountered most often in German Shepherd Dogs.1 Pituitary dwarfism in this breed is inherited in an autosomal recessive fashion2 and is characterized by a combined deficiency of growth hormone (GH), thyroid-stimulating hormone (TSH), prolactin, and the gonadotropins. In contrast, adrenocorticotropic hormone secretion is preserved in these animals.3 The hormone deficiencies can lead to a wide range of clinical manifestations, but the most common ones are marked growth retardation, retention of lanugo or secondary hairs (so-called puppy hair coat) with concurrent lack of primary or guard hairs, and bilateral symmetrical alopecia.1 Recently, we have reported that molecular defects of the LHX3 gene are associated with CPHD.4 LHX3, a member of the LIM homeodomain protein family of DNA-binding transcription factors, is an essential

D

From the Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands (Voorbij, Leegwater, Kooistra). The work was performed at Utrecht University. Corresponding author: A.M.W.Y. Voorbij, Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, PO Box 80154, 3508 TD Utrecht, The Netherlands; e-mail: [email protected].

Submitted April 15, 2014; Revised June 17, 2014; Accepted August 4, 2014. Copyright © 2014 by the American College of Veterinary Internal Medicine DOI: 10.1111/jvim.12448

Abbreviations: 3D BSA CPHD CV FAM gDNA GH GHRH IGF-1 MR PCR RIA TE TR TSH TT4 WI

3-dimensional bovine serum albumin combined pituitary hormone deficiency coefficient of variation fluorescein amidite genomic DNA growth hormone GH-releasing hormone insulin-like growth factor-1 magnetic resonance polymerase chain reaction radioimmunoassay time of echo time of repetition thyroid-stimulating hormone total thyroxine weighted images

regulator of pituitary development.5,6 All but 1 analyzed German Shepherd dwarfs were homozygous for a deletion of a 7 bp sequence in intron 5 of the LHX3 gene, decreasing the intron size to 68 bp. Because of this mutation, the intron becomes too small to be spliced efficiently.4 The 1 exception was compound heterozygous for the 7 bp deletion and an insertion of an asparagine codon in the fragment that codes for the DNA-binding homeodomain of LHX3.4 Congenital dwarfism also is known in Saarloos wolfdogs and Czechoslovakian wolfdogs. Because these breeds are both cross-breeds of German Shepherd Dogs and wolfs, we hypothesized that the dwarfism in these breeds is associated with the same molecular defects found in German Shepherd Dog dwarfs. The

Pituitary Dwarfism in Wolfdogs

aim of the present study was to investigate whether Saarloos wolfdog and Czechoslovakian wolfdog dwarfs have the same genetic basis as do German Shepherd Dog dwarfs. An additional aim was to determine the frequency of carriers of the mutated LHX3 gene among Saarloos and Czechoslovakian wolfdogs used for breeding.

Materials and Methods Dogs and DNA Samples Four Czechoslovakian wolfdogs, 1 male and 3 females, 3–4 months of age, and 2 Saarloos wolfdogs, both female and 1 and 5 months of age, with proportionate dwarfism were presented to the Department of Clinical Sciences of Companion Animals of Utrecht University. Two hundred and thirty-nine clinically healthy Saarloos wolfdogs and 200 clinically healthy Czechoslovakian wolfdogs, intended to be used for breeding, were screened for the mutations of the LHX3 gene associated with pituitary dwarfism in German Shepherd Dogs. Blood samples or buccal swabs were collected and genomic DNA (gDNA) was obtained from the samples using magnetic beads technology and a MSM1 robota according to procedures prescribed by the manufacturer.

Hormone Measurements Plasma GH concentration was measured by a commercially available radioimmunoassay (RIA) for porcine and canine GH.b The intra-assay coefficient of variation (CV) was 7.6% at a plasma concentration of 4.4 lg/L. The sensitivity of the assay was 1 lg/L. Total plasma insulin-like growth factor-1 (IGF-1) concentration was measured with a heterologous RIA validated for the dog,7 after acid-ethanol extraction to remove interfering IGF binding proteins. Plasma IGF was extracted using a mixture of 87.5% (v/v) ethanol and 12.5% 2 M formic acid. Tubes containing 100 lL plasma and 400 lL of the ethanol-formic acid mixture were mixed thoroughly and incubated for 30 minutes at room temperature. After centrifugation for 30 minutes at 5,500 9 g at 4°C, a 50-lL aliquot of the supernatant was diluted 1 : 50 with assay buffer containing 63 mM Na2HPO4 (pH 7.4), 13 mM Na2EDTA, and 0.25% (w/v) bovine serum albumin (BSA). The extraction efficiency was 92.5  5.7%. The intraassay CV was 8.6% at a plasma concentration of 100 lg/L. The sensitivity of the assay was 10 lg/L. IGF-I antiserum AFP4892898 and human IGF-I for iodination were obtained from the National Hormone and Peptide Program.c Plasma total thyroxine (TT4) concentration was measured using a homologous solid-phase, chemiluminescent enzyme immunoassay (Immulite canine total T4d ) according to manufacturer’s instructions and validated for the dog.8 The sensitivity of the assay was 0.16 lg/dL (2 nmol/L). The intra-assay CVs were 13.8% and 8.2% at plasma TT4 concentrations of 0.62 and 1.94 lg/dL (8 and 25 nmol/L), respectively. The interassay CV was 8.5%. Plasma TSH concentration was measured using a homologous solid-phase, 2-site chemiluminescent enzyme immunometric assay (Immulite canine TSHd), according to manufacturer’s instructions.9 The sensitivity of the assay was 0.03 lg/L. The intra-assay CVs were 5.0%, 4.0%, and 3.8% at plasma TSH concentrations of 0.20, 0.50, and 2.6 lg/L, respectively. The interassay CVs were 6.3% and 8.2% at plasma TSH concentrations of 0.16 and 2.8 lg/L, respectively. The upper limit of the reference range for

1771

the plasma TSH concentration in euthyroid dogs in our laboratory is 0.6 lg/L.

Hormone Function Test A GH-releasing hormone (GHRH)-stimulation test was performed by IV administration of 1 lg hGHRHe per kg body weight.10,11 Blood samples for determination of plasma GH concentrations were collected from the jugular vein in chilled EDTA-coated tubes immediately before and 20 minutes after the administration of GHRH.

Diagnostic Imaging Magnetic resonance (MR) images were obtained with a 0.2T open magnet using a 16S multipurpose coil.f Sagittal T2-weighted images (WI) (time of repetition [TR]: 4,455 milliseconds, time of echo [TE]: 117 milliseconds) of the caudal skull were obtained in 3 of the Czechoslovakian wolfdogs and 1 of the Saarloos wolfdogs with proportionate growth retardation. Sequences consisted of 3-mm-thick slices. Flash 3-dimensional (3D)-WI (TR: 34 milliseconds, TE: 12 milliseconds) before and after contrast administration were obtained. All flash 3D series consisted of 1mm-thick T1-weighted slices. Patients were positioned in sternal recumbency.

DNA Analysis Genomic DNA served as a template for PCR amplification. PCR was performed using a primer pair that covered part of exon 5, intron 5, and part of exon 6 of canine LHX3 sequences. The DNA sequence of the forward primer was CCAAGCAGCTGGAGACCTTGAAGAG and the reverse primer had the sequence CTGGACGCTGTCCTTGTCCGAC. The forward primer was labeled with 6-fluorescein amidite (FAM).g The PCR reactions were performed in a 25-lL volume containing 25 ng of gDNA, 1 mM MgSO4, 0.2 mM dNTPs, 0.3 lM of both primers, and 0.5 U Platinum Pfx polymerase in Pfx amplification buffer and PCR enhancer.h The thermo-cycling program consisted of a denaturation step of 2 minutes at 94°C, followed by 35 cycles each of 15 seconds at 94°C, 30 seconds at 61.4°C, and 30 seconds at 68°C. The labeled PCR products were diluted 10-fold in water. One microliter of the dilution was mixed with 15 lL Hi-Di formamide and 0.2 lL of size standard 600-LIZ.i The products were analyzed with the ABI 3130 XL Genetic Analyzer using POP7i and filterset G5i and Genemapper 4.0.i In the case of DNA sequence analysis, the PCR products were treated with Shrimp Alkaline Phosphatasej and Exonuclease Ik and used as a template in a DNA sequencing reaction containing BigDye Terminator v3.1,i according to manufacturer’s instructions. The reaction products were purified with multiscreen 96-well Sephadex 50-gel filtration plates, and analyzed on an ABI 3130XL Genetic Analyzer.i The DNA sequences were aligned using the Seqman program from the Lasergene package of DNASTAR inc.l and compared to the reference canine genome with BLAST software.m

Results All 6 dogs with proportionate dwarfism had retention of their puppy hair coat and a variable degree of alopecia (Fig 1). History, physical examination, and routine blood examinations were otherwise unremarkable. The basal plasma GH concentration was either

1772

Voorbij, Leegwater, and Kooistra

A

B

Fig 2. Sagittal T2-WI of the head of a Czechoslovakian wolfdog dwarf. Note the cyst-like structure in the region of the pituitary gland.

Fig 1. (A) An 8-month-old female intact Czechoslovakian wolfdog with dwarfism. The dog is proportionate and has retention of its puppy hair coat with isolated patches of adult hair. (B) A 1-year-old female intact Saarloos wolfdog with dwarfism. Note the proportionate growth retardation, retention of the puppy hair coat, and alopecia on the hind legs.

immeasurable or very low (median,

Pituitary dwarfism in Saarloos and Czechoslovakian wolfdogs is associated with a mutation in LHX3.

Pituitary dwarfism in German Shepherd Dogs is associated with autosomal recessive inheritance and a mutation in LHX3, resulting in combined pituitary ...
708KB Sizes 0 Downloads 8 Views